Abnormal activity of the dopamine-responsive nervous system has been implicated in a number of motor and behavioral disorders including Parkinson's disease, Huntington's disease, tardive dyskinesia, certain forms of schizophrenia and other dystonias and dyskinesias. Dysfunctions of the dopamine-responsive system may be caused either by a reduced or increased activity of the system or by the inability of the systems to be modulated by a changing external or internal environment.
Dopamine is one of the major catecholamine neurotransmitters in the mammalian brain. Dopamine exerts its effect in part by binding to G protein-coupled dopamine receptors. Pharmacological and molecular biological studies have shown that the dopamine receptor has at least five subtypes, designated D.sub.1-5. The best characterized of these are D.sub.1 and D.sub.2. The D.sub.2 subtype exists in a long and short form, the long form having a larger intracellular loop than the short form. These receptor subtypes appear to be anatomically, biochemically and behaviorally distinct. For example, D.sub.1 and D.sub.2 receptors have different anatomical distributions, in that only D.sub.1 receptors are found in the retina and only D.sub.2 receptors are found in the pituitary, but both D.sub.1 and D.sub.2 are found in the striatum and substantia nigra. D.sub.1 and D.sub.2 receptors are reported to have opposite biochemical effects on adenylate cyclase activity, and stimulation of D.sub.1 and D.sub.2 receptors produces different behavioral responses. See Weiss et al., Neurochemical Pharmacology--A Tribute to B. B. Brody, E. Costa, ed.; Raven Press, Ltd., New York; pp. 149-164 (1989).
Recently, three new subtypes of dopamine receptors have been discovered. On the basis of nucleotide and amino acid sequence homology, D.sub.3 and D.sub.4 have been found to be related to D.sub.2, and D.sub.5 is related to D.sub.1. Hence, the dopamine receptor subtypes may be categorized into two subfamilies, D.sub.1 and D.sub.5 being members of the D.sub.1 subfamily, and D.sub.2, D.sub.3 and D.sub.4 being members of the D.sub.2 subfamily. See Sibley et al., Trends in Pharmacological Sciences, 13: 61-69 (February, 1992).
The dopamine receptor subtypes can be separately and independently modulated through the administration of selective agonists and antagonists. For example, whereas dopamine and apomorphine are agonists of both D.sub.1 and D.sub.2 receptors, compounds such as SKF 38393 (Setler et al., Eur. J. Pharmacol., 50: 419-30, 1978) is a selective agonist of only D.sub.1 and quinpirole (Tsuruta et al., Nature, 292: 463-65, 1981) is a relatively selective agonist for the D.sub.2 receptor. It should be emphasized, however, that the currently available dopaminergic drugs have only a relative selectivity for the various dopamine receptors. Indeed, there are recent reports suggesting that quinpirole may have a higher affinity for D.sub.3 receptors than for D.sub.2 receptors (see Sokoloff et al., Nature, 347: 146-151 (1990)).
It is not surprising, therefore, that the use of specific and nonspecific neuroleptic drugs in the treatment of dopamine-related disorders is contraindicated by the fact that such drugs often produce numerous side effects, presumably due to cross-reactivity with other dopamine receptor subtypes, or even with other classes of neuroreceptors. For example, the therapeutic action of many neuroleptic drugs appears to be due to a blockade of dopamine D.sub.2 receptors. However, patients treated with such drugs often develop tardive dyskinesia, possibly because of the up-regulation of dopamine receptors. A further example is that benzazepines (such as SKF-38393), which are a major D.sub.1 drug class, were recently found to be strongly cross-reactive with the serotonin 5-HT.sub.2 receptor family. Nicklaus et al., J. of Pharmacol. Exp. Ther., 247: 343-48 (1988); Hoyer et al., Eur. J. Pharmacol., 150: 181-84 (1988). Additionally, clozapine is a favored antipsychotic in the treatment of socially withdrawn and treatment-resistent schizophrenics because it does not cause tardive dyskinesia. Clozapine has been found to preferentially bind the D.sub.4 receptor with an affinity ten times higher than to the D.sub.2 or D.sub.3 receptors. Van Tol et al., Nature, 358: 149-152 (1992). However, clozapine treatment results in other side effects that are likely to be related to nonselectivity of the drug.
From the foregoing discussion, it can be seen that a need exists for improved selectivity in dopamine receptor drug classes. However, it is difficult to achieve such selectivity with classical pharmaceutical agents because the available drugs likely act on multiple neurotransmitter receptors and because the modulatory responsiveness of the nervous system to such compounds is not well understood. Therefore, it is difficult to separate the effect of drug cross-reactivity from a natural compensatory response of the nervous system to modulation of a specific receptor.
Molecular biological techniques provide a useful means to develop highly specific receptor modulatory agents. In the case of dopamine receptors, all five subtypes have been cloned from at least one biological source. Sequence information is available for all five subtypes. The availability of such information enables development of receptor-regulatory agents targeted to pre-translational stages of receptor expression. In particular, knowledge of the nucleotide sequence of dopamine receptor genes and messenger RNAs enables the selection and synthesis of antisense molecules capable of binding to critical targets on dopamine receptor mRNAs, thereby inhibiting or modifying translation.
The use of synthetic antisense oligonucleotides for therapeutic purposes was first proposed in 1978 and has been successfully accomplished in vitro and within cultured cells. See Uhlmann et al., Chemical Review, 90: 544-584 (1990). However, successful application of antisense therapy in vivo has been extremely limited. The delivery of antisense oligonucleotides to target cells in vivo is but one obstacle to overcome in developing successful antisense therapeutic agents. Even if biologically significant amounts of antisense molecules reach target cells and bind to selected target sites on mRNA, a subsequent effect on regulation of translation is not guaranteed. Regulation of protein synthesis is highly dependent upon the number of messenger RNA molecules present in the cell that encode a particular protein, as well as the rate of protein synthesis. Furthermore, even if expression of a selected protein can be modulated by an antisense molecule, whether such modulation would have an effect on the associated pathological condition cannot be predicted.
There have been a few reports of successful modulation of various pathological conditions by antisense therapy in rodents. Oligonucleotides antisense to the proto-oncogene c-myc have been administered in vivo to rats to suppress the intimal accumulation of rat carotid arterial smooth muscle cells (Simons et al., Nature 359: 67-70 (1992). Antisense oligonucleotides have also been used in vivo in mice. An 18-nucleotide phosphorothioate oligodeoxynucleotide antisense to sequences encoding the interleukin I (IL-1) receptors, when injected subcutaneously into mice, markedly inhibited the infiltration of neutrophils in response to subsequent injections of IL-1. Burch and Mahan, J. Clin, Invest., 88: 1190-1196 (1991). In another study, repeated injection of antisense oligonucleotide (3.times.0.5 nmol per day) conferred 30-70% protection against a normally fatal infection of encephalitis in mice. See Uhlmann et al., supra at 577.
It can be surmised from the foregoing examples that antisense technology has great potential in vivo therapy for the treatment of disease. However, this potential, to date, remains largely unexplored.